The long-term objective of this proposal is to understand how TRPM8 is modulated by PIRT. The motivation for this proposal is the significant health relevance associated with regulating TRPM8 signaling cascades. TRPM8 is an ion channel that functions as the primary cold sensor in humans. This channel was initially found to be an oncogene that is upregulated in a number of types of cancer and is currently under evaluation as a target of anti-cancer therapy. More recently, TRPM8 has been identified as an attractive target for pharmacological intervention of obesity and chronic pain. It was recently shown that PIRT, a small membrane protein with two transmembrane helices, directly interacts withTRPM8 and alters the response to diverse stimuli. The mechanism of modulation is not currently understood and there is no structural information regarding this complex. In this proposal we will use nuclear magnetic resonance spectroscopy (NMR), electrophysiology, biochemistry, and computational structural biology to generate an experimentally restrained integrative structural model of the TRPM8-PIRT membrane protein complex. Aim 1 will isolate and characterize the regions of the proteins that are required for complex formation using a mix of electrophysiology, NMR binding, and biochemistry studies. This aim will also, for the first time, probe the TRPM8-PIRT complex stoichiometry and identify the PIRT lipid binding site for phosphoinositides. Aim 2 will produce the first structure of the human membrane protein PIRT, a comparative TRPM8 model, and an experimentally restrained integrative structural model of the TRPM8-PIRT complex. Aims 1 and 2 are independent yet cooperative in nature: the results from Aim 1 can be used to guide the integrative structural biology in Aim 2 and the output from Aim 2 can be used to generate hypotheses and instruct the isolation of the complex functional determinants. We have generated significant preliminary electrophysiology, NMR, and computational data that suggest these aims are feasible during the timeframe of this proposal. For Aim 1, both NMR and electrophysiology data that suggest the TRPM8 voltage-sensing domain is key to PIRT modulation and we have isolated specific residues in TRPM8 that are key to functional modulation. Preliminary data for Aim 2 suggest that NMR structural studies of PIRT will result in the first structure of this protein. The proposed structural and functional analysisof PIRT modulation of TRPM8 will increase our understanding of how to regulate this channel. Moreover, understanding the structure and function of PIRT may provide an alternative therapeutic pathway to modulate TRP channel function with potentially fewer off target effects.